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US 10,590,415 B2Granted
Engineered nucleic acid-targeting nucleic acids
AssigneeCeribou Biosciences, Inc.
InventorsPaul Daniel Donohoue, Andrew Paul May
Life of this patent
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Abstract
The present disclosure provides engineered Class 2 CRISPR-Cas-associated discontinuous first-stem nucleic-acid targeting nucleic acids, nucleoprotein complexes comprising these nucleic acids, and compositions thereof. Nucleic acid sequences encoding the Class 2 CRISPR-Cas-associated discontinuous first-stem nucleic-acid targeting nucleic acids, as well as expression cassettes, vectors and cells comprising such nucleic acid sequences, are described. Also, methods are disclosed for making and using the Class 2 CRISPR-Cas-associated discontinuous first-stem nucleic-acid targeting nucleic acids, nucleoprotein complexes comprising such nucleic acids, and compositions thereof.
Claims20
A method of binding a nucleic acid target sequence, comprising:
- contacting the nucleic acid target sequence with a Class 2 Type II CRISPR-Cas9-nucleoprotein composition comprising,
- a CRISPR Type II Cas9 protein, and
- a Class 2 Type II CRISPR-Cas9-associated discontinuous first-stem nucleic-acid targeting nucleic acid (dfs-NATNA) composition, comprising,
- a first Class 2 Type II CRISPR-Cas9-associated discontinuous first-stem single-strand polynucleotide (dfs1-PN) comprising, in a 5′ to 3′ direction,
- a fragment nucleotide sequence 1 comprising a 5′ terminus of the dfs1-PN and at least 2 nucleotides,
- a bulge element nucleotide sequence II,
- an upper stem element nucleotide sequence II,
- a first linker nucleotide sequence,
- an upper stem element nucleotide sequence I,
- a bulge element nucleotide sequence I,
- a lower stem element nucleotide sequence I,
- a second linker nucleotide sequence,
- a nexus nucleotide sequence,
- a third linker nucleotide sequence, and
- a 3′ hairpin element;
- and
- a second Class 2 Type II CRISPR-Cas9-associated discontinuous first-stem single-strand polynucleotide (dfs2-PN) comprising, in a 5′ to 3′ direction,
- a nucleic acid target binding sequence, and
- a fragment nucleotide sequence 2 comprising at least 2 nucleotides and a 3′ terminus of the dfs2-PN,
- wherein the upper stem element nucleotide sequence I and the upper stem element nucleotide sequence II base-pair hydrogen bond to form an upper stem element;
- wherein the bulge element nucleotide sequence I and the bulge element nucleotide sequence II form a bulge element;
- and
- wherein at least 2 nucleotides of the fragment nucleotide sequence 1 form 2 pairs of hydrogen-bonded nucleotides with the lower stem element nucleotide sequence I and at least 2 nucleotides of the fragment nucleotide sequence 2 form 2 pairs of hydrogen-bonded nucleotides with the lower stem element nucleotide sequence I to form a lower stem element;
- or
- a first Class 2 Type II CRISPR-Cas9-associated discontinuous first-stem single-strand polynucleotide (dfs1-PN) comprising, in a 5′ to 3′ direction,
- a fragment nucleotide sequence 1 comprising a 5′ terminus of the dfs1-PN and at least 2 nucleotides,
- a first linker nucleotide sequence,
- a nexus nucleotide sequence,
- a second linker nucleotide sequence, and
- a 3′ hairpin element;
- and
- a second Class 2 Type II CRISPR-Cas9-associated discontinuous first-stem single-strand polynucleotide (dfs2-PN) comprising, in a 5′ to 3′ direction,
- a nucleic acid target binding sequence,
- a lower stem element nucleotide sequence II,
- a bulge element nucleotide sequence II,
- an upper stem element nucleotide sequence II,
- a third linker nucleotide sequence,
- an upper stem element nucleotide sequence I,
- a bulge element nucleotide sequence I, and
- a fragment nucleotide sequence 2 comprising at least 2 nucleotides and a 3′ terminus of the dfs2-PN,
- wherein the upper stem element nucleotide sequence I and the upper stem element nucleotide sequence II base-pair hydrogen bond to form an upper stem element;
- wherein the bulge element nucleotide sequence I and the bulge element nucleotide sequence II form a bulge element;
- and
- wherein at least 2 nucleotides of the fragment nucleotide sequence 1 form 2 pairs of hydrogen-bonded nucleotides with the lower stem element nucleotide sequence II and at least 2 nucleotides of the fragment nucleotide sequence 2 form 2 pairs of hydrogen-bonded nucleotides with the lower stem element nucleotide sequence II to form a lower stem element;
- thereby facilitating binding of the Class2 Type II CRISPR-Cas9 nucleoprotein composition to the nucleic acid target sequence.
42 more elements
Dependencies are inferred from the claim text.